Protein fragment complementation assays for the detection of biological or drug interactions
US6428951B1 · kind B1 · utility
Assignee
Inventors
Key dates
| Filing date | Feb 7, 2000 |
| Grant date | Aug 6, 2002 |
| Priority date | — |
| Expiry date | Feb 7, 2020 |
Classification
- Technology area (CPC Y)Emerging Cross-Sectional Technologies
- CPC primaryY02A90/10
- WIPO fieldMeasurement
- WIPO sectorInstruments
Abstract
We describe a strategy for designing and implementing protein-fragment complementation assays (PCAs) to detect biomolecular interactions in vivo and in vitro. The design, implementation and broad applications of this strategy are illustrated with a large number of enzymes with particular detail provided for the example of murine dihydrofolate reductase (DHFR). Fusion peptides consisting of N- and C-terminal fragments of murine DHFR fused to-GCN4 leucine zipper sequences were coexpressed in Escherichia coli grown in minimal medium, where the endogenous DHFR activity was inhibited with trimethoprim. Coexpression of the complementary fusion products restored colony formation. Survival only occurred when both DHFR fragments were present and contained leucine-zipper forming sequences, demonstrating that reconstitution of enzyme activity requires assistance of leucine zipper formation. DHFR fragment-interface point mutants of increasing severity (lie to Val, Ala and Gly) resulted in a sequential increase in E. coli doubling times illustrating the successful DHFR fragment reassembly rather than non-specific interactions between fragments. This assay could be used to study equilibrium and …
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